4-dedimethylaminatetracycline and 5a, 6-anhydro derivatives thereof

ABSTRACT

4-Dedimethylaminotetracyclines and the corresponding 5a,6anhydroderivatives having an oxo, hydroxy, substituted imino, amino or substituted amino group other than dimethylamino at the C.4-position useful as antimicrobial agents. The 4-oxo-4dedimethylaminotetracyclines are prepared by oxidation of tetracyclines with a hydrocarbon dicarboxylic acid haloimide and then converted by reduction to the corresponding 4-hydroxy compounds or by reaction with a primary amine to 4-substituted imino derivatives. The latter compounds are reduced to 4-amino derivatives which are reductively methylated or alkylated to 4substituted-4-dedimethylaminotetracyclines. Dehydration of the tetracycline compounds affords the corresponding 5a,6-anhydro compounds.

United States Patent [72] Inventors Robert K. Blnckwood Gales Ferry; Charles R. Stephens, Jr., East Lyme, both of Conn.

[21] Appl. No. 667,357

[22] Filed Sept. 13, 1967 Continuation-impart of Ser. No. 360,435, Apr. 16, 1964, abandoned, which is a continuation-in-part of Ser. No. 247, 874, Dec. 28, 1962, abandoned.

Nov. 23, 1971 New York, N.Y.

Patented Assignee [54] 4-DEDIMETHYLAMINATETRACYCLINE AND 5A,

6-ANl-lYDRO DERIVATIVES THEREOF 5 Claims, No Drawings [56] References Cited UNITED STATES PATENTS 3,159,675 12/1964 Esse et a1. 260/559 AT Primary Examiner-Alex Mazel Assistant Examiner-Anne Mariet Tighe Attorney-Connolly and Hutz ABSTRACT: 4-Dedimethylaminotetracyclines and the corresponding 5a,6-anhydroderivatives having an oxo, hydroxy, substituted imino, amino or substituted amino group other than dimethylamino at the C'4-position useful as antimicrobial agents. The 4-oxo-4-dedimethylaminotetracyclines are prepared by oxidation of tetracyclines with a hydrocarbon dicarboxylic acid haloimide and then converted by reduction to the corresponding 4-hydroxy compounds or by reaction with a primary amine to 4-substituted imino derivatives. The latter compounds are reduced to 4-amino derivatives which are reductively methylated or alkylated to 4-substituted-4- dedimethylaminotetracyclines. Dehydration of the tetracycline compounds affords the corresponding 5a,6-anhydro compounds.

annonvmmvmmmnnmcvcmmsme YX H on Y x on .NR;

ANHYDRODERIVA'I'IVESTHEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of copending application Ser. No. 360,435, filed Apr. 16, 1964, now abandoned which in turn is a continuation-in-part of application N313: Y X on Set. No. 247,874, filed Dec. 28, 1962,now abandoned. Y f A I on -on BACKGROUND OF THE INVENTION OHH VCONH;

This invention is concerned with new and useful antimicrobial agents, and more particularly with novel 4-oxo-4- dedimethylamino derivatives of the tetracycline antibiotics and certain novel products obtained therefrom.

The tetracycline antibiotics comprise a group of biologically Y Y CH3 0 X NR, I II I I active hydronaphthacene derivatives having the following es- A sential structural features: T 0H 0 ON H 0 ONE I I z OH on d d I III 1654 1101100 8 a, W 1 2 C ONHI wherein R is selected from the group consisting of hydrogen, 6 A A) y) benzyl, phenethyl, cyclohexyl and alkyl of from two to six car- H H bon atoms; R; is selected from the group consisting of hydrogen, alkyl of from two to six carbon atoms and hydroxyalkyl of from two to four carbon atoms; R is selected from Among the biologically active members of this group are the the 8 P cmststing 05 NHCONH W m following; NHCSNH 4(B-hydroxyethyl)piperazino, and NR,R wherein R is selected from the group consisting of hydrogen, alkyl of from one to six carbon atoms, phenyl, benzyl and cyclohexyl;

R is selected from the group consisting of hydrogen, alkyl of Subnituents Common Name from one to SIX carbon atoms which may be straight-chained LNCHUGoHMHMhOH Tetracycline 40 or branched-chained, phenyl, and benzyl; 4-N(CH,),,5-Ol-l,6-OH,6-CH,.I2a-OH S-Oxytetracycline Z 15 selected from the group consisting of hydrogen, chloro r: asses-2:22; and

I eoxy- -oxytetnu:yc inc L 5 @Deomemcwim X is selected from the group consisting of methyl and 4-N(CH,),,l2a-OH 6-Deoxy-fi-demethyltetracycline hydrogen, the Proviso that x methyl when 2 4-N(CH,),.6 OH,6-CH,,7-Br, l Za-OH 7-Bromotetracycline hydrogen; "mcmheomrcuhnn Y is selected from the group consisting of hydrogen and 4-N(CH ,6-OH.l2a-0H 6-Demethyltetracycline 4-N(CH:):,6-(=Cl-l,),l2a-0H 6-Methyleneletracycline 4-N(CH;),,6-(=CH,).IIn-CLI2a-OH llrChloro-6-methylenetetracycline A preferred member of this class of derivatives IS 4-oxo-4- dedimethylaminotetracycline-4,6-hemiketal, having the struc- 4-N(CH,),.$-OH.6-(#Il-l,).l la'cl, lla-Chloro-6-methylene-5- t I T h .k W n] f t t m0" omemcycfim ure e emi e c ures o is mven on exis in equilibrium with the equivalent nonketallzed form, II.

CH; 0 SUMMARY OF THE INVENTION The novel compounds of the present invention have the general formulas:

H O I Y X 0 OH Y H3O OH NRiRa V I and wherever 4,6-hemiketals are referred to herein and in the OH OH appended claims, the term is intended to embrace the cor OH OH responding nonketalized form as well. C ONHZ The reaction sequences employed in the preparation of the 1 z 1 g a 1| compounds of the present invention are exemplified in the fol- OH OH O lowing flowsheet using tetracycline as the reactant.

I ll 0H 0 DETAILED DESCRIPTION OF THE INVENTION IV V represent reductions of the 4-oxo group to 4- hydroxy In this reaction sequence R,, R, and R, are as defined and b 7 H-A or 11-13 [V represent dehydration reactions to form 5a,6-anhydro compounds. I ll-A represents an oxidation reaction. II-A or [1-8 V] and IV VIII represent reaction with II-A or [1-8 Ill and carbonyl group reagents.

V] VII and VIII X represent reduction reactions.

VIl XI and X IX represent alkylations.

The oxidation reaction to form a 4,6-hemiketal may be effected under acid conditions by treating a compound of the formula: 5

-CONH, 6H 82 1% ll wherein X and Z have the above significance with a hydrocarbon dicarboxylic acid haloimide such as N-chlorsuccinimide, preferably in aqueous media and at pH below 2. Dilute mineral acid such as dilute hydrochloric acid is a preferred medium. The reaction is readily carried out at room temperature, and the product may be recovered by filtration and purified by such methods as solvent extraction, treatment with activated carbon, and precipitation in the form of a metal salt, e.g. an alkali metal or alkaline earth metal salt. The equilibrium between the hemiketal and the nonketalized form is shifted in favor of the herniketal in the crystalline state and in acid media; under basic conditions the equilibrium appears to shift toward the nonketalized form, although under extremely basic conditions this is accompanied by degradation. Thus, prolonged treatment with liquid ammonia or aqueous potassium hydroxide at about pH 12 leads to the formation of compounds such as the following.

Application of the described N-chlorsuccinimide treatment to l1a-halotetracycline-6,l2-hemiketals having the following formula:

1| X 0 N(CH;),

I 0 ONE, OH i Z 0 H wherein X, Y and Z are as defined above, provides 4-oxo-4- 6o dedimethylaminol la-halotetracycline-4,6-hemiketals; 6-

deoxy-fi demethyltetracycline and lla-chloro-G- methylenetetracycline yield similar nonbasic products under these conditions.

The reduction reactions to produce a 4-hydroxy group may be carried out at room temperature by various means, including treatment with sodium hydrosulfite, zinc dust in dilute mineral acid or aqueous acetic acid, or mild catalytic hydrogenation. A preferred procedure entails treatment with an alkali metal hydrosulfite such as sodium hydrosulfite in a suitable solvent, e.g. aqueous alcohol, followed by recovery and purification by such methods as solvent extraction and recrystallization.

The dehydration reactions to produce 5a,6-anhydro compounds are effected by treatment insolution with mineral acid the reaction mixture where solubility is limited, or altematively by evaporation of the reaction medium.

The 4-hydroxy-5a,6-anhydrotetracyclines of the present invention are characterized by excellent stability and exhibit surprisingly high biological activity against various micro-organisms including antibiotic-resistant strains. For example, 4- hydroxy-4-dedimethylamino-5a,6-anhydrotetracycline, when tested in vitro by the usual serial dilution technique, exhibits the following minimum inhibitory concentrations against various disease-causing micro-organisms:

M.I.C. Species (mc .lrnl.)

Mirror'rxr'us pyogenes var. aureur l2.5 Micrococru: pyogene: var. aureur 400' 25 Slreplocoa'u: pyugene: 3. l 2 Slrepwmcau pyugrne: 98 L56 Slreplomrnufaerali: l2.5 Diplocoaus pneumonia: 25 Eryripelolhrix rhuriupalhiae 6.25 Escherichia coli 25 Salmonella lypharn l2.5 Kle siella pneumoniae 25 Hemophilu: influenza: 50

Antibiotic resistant strain As will be seen from the above data, these novel anhydrotetracyclines are useful in therapy, in agriculture, and in veterinary practice both therapeutically and as growth stimulants. In addition, they may be employed as disinfectants and bacteriostatic agents, in industrial ferrnentations to prevent contamination by sensitive organisms, and in tissue culture, e.g. for vaccine production. They are likewise useful in separating and; classifying mixtures of micro-organisms for biological research and medical diagnostic purposes.

The 4,6-hemiketal and the 4-oxo derivatives of this invention are useful intermediates for the introduction of new substituents at the 4 position of the tetracycline and anhydrotetracycline compounds. Thus, they may be subjected to reductive amination in the presence of reducing agents such as formic acid or its derivatives, or hydrogen together with noble metal catalysts, to introduce new 4-amino substituents. They may be reacted with various carbonyl group reagents, that is, reagents derived from ammonia which react rather generally with the carbonyl group of aldehyde: and ketones. Typical of such reagents are those having the fonnula ,NR wherein R, is as defined above. Representative compounds are hydroxylamine, hydrazine, semicarbazide, phenylhydrazine, thiosemicarbazide, monoand unsymmetrically disubstituted hydrazines.

The hydrazine and other carbonyl group reagents may be used as their acid salts or as the free bases. The lower alcohols are the preferred solvents, particularly when using the free bases as reagent. In certain solvents, ether for example, simple salt formation rather than reaction at the keto group occurs. However, since a wide variety of solvents are operative, the choice of solvent is readily determined by experiment on a small scale. The lower alcohols, methyl, ethyl, isopropyl, etc., or combinations thereof, are favored.

The resulting oximes, hydrazones, semicarbazones and thiosemicarbazones may be reduced to the corresponding saturated compounds under appropriate conditions, or to the 4-amino-4-dedimethylaminotetracyclines and 4-arnino-4- dedimethylaminoanhydrotetracyclines.

Reductive amination may be conducted with ammonia and primary amines includingmonoalkanolamines, alkylamines of from one to six carbon atoms, benzylamine, B- phenethylamine, aniline and cyclohexylamine. The reaction is conducted in a suitable reaction-inert solvent system such as dimethylformamide at a temperature of from about 20 C. to about 50 C. Low pressures, that is, pressures of from about 1 atmosphere to about 150 atmospheres are preferred mostly for the sake of convenience. Higher pressures can be used but appear to offer no advantages. Platinum oxide is favored as catalyst since it produces satisfactory yields. Other catalysts such as Raney nickel may also be used. An excess of the ammonia or amine is used. The addition of the hydrochloride salt of ammonia or of the amine improves the yield in many instances.

It has been found advantageous, in order to minimize decomposition of the reactants and products, to use the tetracycline -4,6-hemiketal compounds in the fonn of a chelate of a polyvalent metal ion such as magnesium, calcium, zinc, copper, cobalt, nickel, manganese and cadmium. The metal chelate may be formed in situ, preferably by addition of a suitable solvent soluble salt, e.g. the polyvalent metal chloride, acetate, nitrate, etc., or may be preformed. A 1:1 molar ratio of polyvalent metal ion to tetracycline-4,6-hemiketal is preferred. The same considerations apply to the 4-oxo-5a,6- anhydrotetracyclines. The reductive amination product is regenerated from its metal chelate by known methods, e.g. acid treatment to decompose the chelate, ion-exchange treatment or precipitation of the chelated metal as an insoluble salt, such as the sulfide or oxalate.

Reductive amination of 4-oxo-4-dedimethylaminotetracycline-4,6-hemiketal with ammonium hydroxide in the presence of a polyvalent metal ion, e.g. magnesium, produces a mixture of 4-epiand 4-norrnal-amino4- dedimethylaminotetracycline in which the 4-epi compound predominates. Methylamine under similar conditions produces predominately the 4-epi compound, 4-epi-4-methylamino-4-dedimethylaminotetracycline. The other amines mentioned above also produce the 4-epi compound as major product.

Chemical and catalytic reduction of the compounds bearing a NR, group at the 4-position to 4-amino-4- dedimethylaminotetracyclines also produces a mixture of the 4-epiand 4-normal-amino-4-dedimethylaminotetracyclines in which the 4-epi compound predominates. The 4-normal compounds exhibit much greater antibacterial activity than do the 4-epi compounds. The formulas used herein are intended to embrace the epiand normal configurations at the C.4 position.

The 4-amino-4-dedimethylaminotetracycline (the 4-normal compound referred to above) is isolated from the mixture containing the 4-epi compound by warming the mixture in mild basic solution, e.g. aqueous sodium or potassium bicarbonate, whereby the 4-epi compound is degraded. The normal 4-aminotetracycline is recovered by suitable means as by solvent extraction. This method is applicable to the isolation of the 4-normal-monosubstituted-amino-4- dedimethylaminotetracyclines in general. The noval 4-amino- 4-dedimethylaminotetracyclines and anhydrotetracyclines of this invention (epi and normal configuration) may be alkylated in a suitable solvent system (tetrahydrofuran or acetone) by means of' appropriate agents such as alkyl, alicyclic and aralkyl iodides and dialkyl sulfates. The use of an acid acceptor (inorganic or organic base) or acid scavenger (propylene oxide) is required to promote dialkylation. The use of excess alkyl, alicyclic or aralkyl iodide plus acid acceptor or scavenger in any of these alkylations leads to betaine formation.

They may also be reductively alkylated using an aldehyde or ketone in the presence of a reducing agent such as hydrogen and a catalyst, e.g. Raney nickel or platinum. Lower alkanols, especially methanol or ethanol are favored solvent systems. Hydrogen pressures of from about 1 to 150 atmospheres and temperatures of from about 20-80 C. are suitable.

The 4-hydroxy-4-dedimethylaminotetracyclines, while surprisingly possessing negligible in vitro biological activity against a variety of Gram-positive and Gram-negative microorganisms tested, are useful intermediates, and may be converted to readily replaceable sulfonic esters such as the tosylates, which may in turn be employed in nucleophilic replacement reactions, e.g. with amines, mercaptans, cyanides and the like to introduce C. substituents.

The present invention embraces all metal salts of the new products and all acid addition salts of the new basic products described herein. The well-known procedures for preparing salts of tetracycline compounds are applicable here and are illustrated by examples appearing hereinafter. Such salts may be formed with pharmaceutically acceptable and pharmaceutically unacceptable metals and acids. By pharmaceutically acceptable is meant those salt-forming metal ions and acids which do not substantially increase the toxicity of the tetracycline compound. Of particular value in the case of the therapeutically active tetracyclines are the pharmaceutically acceptable metal and acid addition salts. In the case of the compounds herein described which are primarily useful & intermediates, the most useful salts are the alkali and alkalineearth metal salts.

The pharmaceutically acceptable metals include sodium, potassium and alkaline-earth metals of atomic number up to and including 20, i.e. magnesium and calcium, and additionally aluminum, zinc, iron and manganese, among others. A

Of course, the metal salts include complex salts, i.e. metal chelates, which are well recognized in the tetracycline art. The pharmaceutically unacceptable metal salts embrace most commonly salts of lithium and of alkaline-earth metals of atomic number greater than 20, i.e. barium and strontium, which are useful for isolating and purifying the compounds. The new substances also form salts with amines of sufficient basicity.

The pharmaceutically acceptable acid addition salts are of particular value in therapy. These include salts of mineral acids such as hydrochloric, hydriodic, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, as well as salts or organic acids such as tartaric, acetic, citric, malic, benzoic, glycollic, gluconic, gulonic, succinic, arylsulfonic, e.g. p-toluenesulfonic acids, and the like. The pharmaceutically unacceptable acid addition salts, while not useful for therapy, are valuable for isolation and purification of the new substances. Further, they are useful for the preparation of pharmaceutically acceptable salts. Of this group, the more common salts include those formed with hydrofluoric and perchloric acids. Hydrofluoride salts are particularly useful for the preparation of the preparation of pharmaceutically acceptable salts, e.g. the hydrochlorides, by solution in hydrochloric acid and crystallization of the hydrochloride salt formed. The perchloric acid salts are useful for purification and crystallation of the new products.

The herein described compounds are useful as complexing agents for polyvalent metal ions. The complexes are usually quite soluble in organic solvents and are useful for purposes wherein metal ion contamination is a problem; e.g. stabilizers in fatty acids, waxes, hydrocarbons, biological experimentation, metal extraction, and as metal carriers.

The following examples are provided for illustrative purposes.

EXAMPLE I 4-Oxo-4-Dedimethylaminotetracycline-4,6-Hemiketal A. The tetracycline hydrochloride, g., is dissolved with stirring in a mixture of 14 ml. concentrated hydrochloric acid and 3,500 ml. water. To this is added49 g. N-chlorsuccinimide. After stirring for 1 hour the reaction mixture is filtered and the solid is washed thoroughly with water and air-dried to obtain 47.1 g. of the desired product.

For purification, 21.0 g. of this material is dissolved with stirring at room temperature in 420 ml. water plus 1,300 ml. ether. The water layer is discarded and the ether is washed with 5x130 ml. portions of water. After drying over sodium sulfate, the ether solution is evaporated to an oil. About 300 ml. water is added and the mixture stirred for 2 hours and filtered. Water-washing and air-drying yield 17.5 g. of partially purified solid.

Further purification is achieved by conversion to the potassium salt in the following manner: 1.0 g. of this product is dissolved in 4.0 ml. methanol at room temperature with stirring. This solution is treated with Darco G-60 activated carbon, filtered and the filtrate treated with 0.40 ml. N potassium hydroxide aqueous solution (1 millimole). This mixture is stirred for minutes at room temperature, filtered, and the recovered solid is methanol-washed and dried under vacuum.

Analysis Found: C, 51.96;l-1, 3.83; N, 3.22; K, 8.18; E 0, 1.71

Calcd. for

C H O NK05H- O: C, 52.0%; H, 3.8; N, 3.2; K, 8.2; H 0, 1 7

This product exhibits ultraviolet absorption maxima in 0.01 N methanolic HCl at 268 and 346 mu, 1oge4.40 and 3.72, and an infrared absorption maximum in potassium bromide at 5.7 ,1. In 0.01 N methanolic NaOl-l decomposition appears to occur, but fresh solutions exhibit ultraviolet absorption maxima at about 265, 319 and 372 mg.

The sodium salt is prepared in the same manner, substituting sodium hydroxide for potassium hydroxide.

B. An attempt to prepare the title compound by air oxidation of tetracycline produced no detectable amount of the compound.

A solution of tetracycline (l g.) in dimethylformamide (30 ml.) is exposed to air at room temperature for ten days then evaporated to dryness under reduced pressure. The residue is slurried in ml. of 0.1 N hydorchloric acid, and the insoluble material collected by filtration (0.66 g.

Treatment of the crude product with sodium hydrosulfite failed to produce the fluorescent 4-hydroxy compound in contrast to the 4-oxo compound produced in method A. It is concluded that no 4oxo-4-dedimethylaminotetracyline-4,6- hemiketal is produced by air oxidation.

. Example 11 4-0xol l a-Chloro-4-Dedimethylaminotetracyc1ine-4,6-

l-lemiltetal l 1a-Chlortetracycline-6-, l2-hemiketal, 20.5 g., is combined with a solution of 6.3 ml. concentrated hydrochloric acid in 1025 ml. water and 14.5 g. N-chlorsuccinimide is added. After stirring 1 hour at room temperature the reaction mixture is filtered and the 10.8 g. of solid product waterwashed and dried.

This substance is further purified by extraction with a mixture of 255 ml. water plus 631 ml. ether. The water layer is then reextraeted with 350 ml. ether, and the ether layers are combined, washed with 3X50 ml. volumes of water and 2X50 ml. volumes of saturated brine. The ether is evaporated. The resulting crystalline solid is slurried with 15 ml. methanol, and 6.2 g. solid product recovered by filtration. lt exhibits infrared absorption maxima at 5.77, 6.02, 6.32 and 6.55 p; ultraviolet absorption maxima occur at 258 and 343 my. in 0.01 N methanolic HCl, loge 4.42 and 3.68, respectively.

Analysis Found: C, 53.0; H. 3.7; N, 2.8; CI, 8.0 Ca1cd.for:C,,,l-1,.O,NCI: c, 53.4; H, 3.6; N, 1|; CI, 7.9

The starting compound for this reaction is prepared in the following manner:

To a solution of 2.2 g. anhydrous tetracycline in 25 ml. ethylene glycol dimethylether is added 800 mg. N-chlorsuccinimide with stirring to dissolve the reagent. The mixture is allowed to stand for 7 minutes and then diluted with 25 ml. water. The produce, 873 mg., crystallizes as white needles. lnfrared analysis shows no carbonyl bands between 5 and 6 .1..

It is purified further by stirring for 20 minutes in 15 ml. methanol. lnfrared absorption maxima are exhibited at the following wave lengths when measured in a KBr pellet at 1 percent concentration: 2.93, 5.70, 6.02, 6 32, 655, 7.15, 7.25, 7.47, 7.58, 7.87, 8.08, 8.19, 8.41, 8.50, 9.05, 9.52, 9.82, 10.04, 10.41, 10.95,1l.54,11.77, 12.12,12.32, 12.50,12.80, 13.28, 13.47 .1.. The ratio ofE,'f,,, values measured in 0.01 N 5 methanolic-HCI at 261 and 345 ml. is 5.6.

By means of this procedure the following 4-oxo-4- dedimethylaminotetracycline-4,6-hemiketals are prepared from the appropriate tetracycline compound.

The llafluoro and chloro-tetracycline-6,lZ-hemiketal reactants are prepared according to the procedure of US. Pat. No. 3,109,007, issued Oct. 29, 1963.

Example 111 4-Hydroxy-4-Dedimethylaminotetracyeline Method A. The crude product of example 1, 25.0 g., is dis solved with stirring in 312.5 ml. methanol and to this is added a solution of 10 g. sodium hydrosulfite in 225 ml. water. Afier stirring at room temperature for minutes, the reaction mixture is combined with 250 ml. water and 1250 ml. ether. The ether extract is separated and the aqueous layer reextracted with another 1250 ml. fresh ether. The two ether layers are washed with water, for 1 hour, filtered and washed thoroughly with water to obtain 19.1 g. of the desired product.

For purification, 1.0 g. of this material is dissolved in 60 ml. ethylene dichloride with heating, and the solution is filtered. Upon cooling the filtrate crystallizes and the 200 mg. of crystalline product is separated by filtration. A second 200 mg. crop of light yellow-green crystals is obtained from the mother liquor. The product exhibits ultraviolet absorption maxima in 0.01 N methanolic HCl at 257 and 362 mp, loge 4.23 and 4.20; in 0.01 N methanolic NaOH at 246, 264 and 55 377 my. and in 0.01 N mg. Cl, at 259, 339 and 373 mp.

Found: Calcd. for C, H NO.:

Method B. The title product of Example 11 (500 mg.) is dissolved in 25 ml. ethyl acetate and hydrogenated over 10 percent Pd/C (50 mg. at 1 atmosphere hydrogen and 25 C. until 2 molar equivalents of hydrogen are taken up The catalyst is removed by filtration and the solvent stripped to give a yield of Method C. Five hundred mg. of the title product of example 11, zinc dust (200 mg.) and 5 ml. of 50 percent aqueous acetic acid are stirred vigorously at room temperature for 15 minutes. The mixture is then filtered and the filtrate freezedried Treatment of the residue with dilute hydrochloric acid yields free 4-hydroxy-4-dedimethylaminotetracycline.

The remaining products of example 11 are reduced to the correspondingv 4-hydroxy-4-dedimethy1aminotetracyelines having the formula:

'fx OH OH CONH,

I II II OH 0 0 wherein Y and X have the values listed in example ll by the above procedures. The l la-halogenated products of Example ll are reduced by methods B and C. To avoid removal of the 7- chloro group hydrogenation is terminated when 2 equivalents of hydrogen are consumed. Alternatively, rhodium is used as catalyst. The zinc dust-acetic acid system of method C is replaced by zinc dust-mineral acid in the case of the llafluoro compounds for more etficient reaction.

Example lV 4-Hydroxy-4-Dedimethylamino-5a,6-Anhydrotetracycline Method A. The title product of example lll, 500 mg., is dissolved at room temperature in ml. methanol and the solution is filtered. To the filtrate is added 3 ml. concentrated hydrochloric acid and the mixture is heated on a steam bath until precipitation begins (about 3-5 minutes). The reaction mixture is then permitted to cool to room temperature and the desired solid product separated by filtration. The yield is 400 mg. of material exhibiting ultraviolet absorption maxima at 272 and 423 my. in 0.01 methanolic HCl.

Method B. The title product of example Ill, 500 mg., is dissolved in 5.0 ml. hot acetone; 10 g. paratoluenesulfonic acid is then added and dissolved with further heating. The reaction mixture is treated with Darco KB activated carbon and filtered. The filtrate is then evaporated to an oil, which crystallizes. The orange crystalline solid is recovered by filtration, washed with a little acetone, and dried. The product exhibits R, values of 0.9 and 0.95, respectively, in the following systems:

Mobile Phase Stationary Phase :3 by volume toluenezpyridine 20:!013 by volume nitromethsne:

chloroformcpyridine pH 4.2 aqueous buffer pH 3.5aqueous buller Elemental Analysis Ultraviolet absorption maxima are observed at 222, 262, 310

Example V 4-0xo-4-Dedimethylamino-5a,6-Anhydrotetracycline The product of example I, l00 mg., is stirred in an ice-water bath with 2.0 ml. liquid hydrogen fluoride for 95 minutes. The hydrogen fluoride is then evaporated in a stream of nitrogen and the residue is stirred at room temperature with about 19 ml. dry ether for 45 minutes and then filtered and dried to obtain 37 mg. of the desired solid product. This substance is further convertible to the product of example IV by treatment with sodium hydrosulfite according to the procedure of example III. This reactivity is readily demonstrated by treating a l mg./ml. solution of the title product in lzl methanolzwater with 10 mg./ml. sodium hydrosulfite; this solution may then be subjected to partition chromatography in 20:3 by volume toluene: pyridine on Whatman chromatographic paper saturated with pH 4.2 aqueous Mcllvaine bufier. Whereas 4-oxo- 4-dedimethylamino-5a,6-anhydrotetracycline in the absence of sodium hydrosulfite exhibits an orange spot at the origin in this system, in the presence of the sodium hydrosulfite a new spot appears corresponding to 4-hydroxy-4- dedimethylamino-5a,6-anhydrotetracycline at an R, value of about 0.9.

Example Vl 4-Hydrazono-4-Dedimethylaminotetracycline A. A mixture of 15 g. of 4-oxo-4-dedimethylaminotetracycline-4,6-hemiketal, 61 ml. of methanol, 30 g. of potassium bicarbonate, and 5.7 g. of powdered hydrazine dihydrochloride is stirred for 30 minutes at room temperature. The reaction mixture is then filtered and washed with methanol. 900 ml. of water containing 6 ml. of concentrated hydrochloric acid is slowly added to the mother liquor with stirring and the mixture allowed to stand. The product which crystallizes over a 45minute period is recovered by filtration, washed with water and dried to give 7.6 g. of product. It is purified by stirring for 2 hours in 3A ethanol and refiltered.

An analytical sample is obtained by recrystallization from tetrahydrofuran-water. The crystalline product is dried in The product exhibits ultraviolet absorption maxima in 0.0l N methanolic-HCl at 264 and 335 mu, loge 4.l6 and 4.53, respectively. In 0.01 N methanolic-sodium hydroxide ultraviolet absorption maxima are exhibited at 26l, 323 and 373 mp, loge 4.33, 4.25 and 4.29, respectively.

B. Crude 4-oxo-4dedimethylaminotetracycline-4,6- hemiketal (417 mg.) is dissolved in 3 ml. of 3A-ethanol and 0.06 ml. of hydrazine hydrate in 3.5 ml. of 3A-ethanol added over a l5-minute period with stirring. The mixture is stirred an additional l5 minutes, the crystalline 4-hydrazono product filtered, washed with 3A-ethanol and dried.

Substitution of hydrazine hydrate or its dihydrochloride salt of methods 8 and A by the appropriate carbonyl reagent produces the following 4-substituted-4-dedimethylaminotetracyclines. For convenience only the R group of the C-4 substituent (=NR,) is given.

The 4-oxotetracyclines of example II are likewise treated pounds bearing the=NR, group at C4.

C. Crude- 4-oxo-l la-chloro-4-dedimethylaminotetracycline-4,6-hemiketal (450 mg.) is dissolved in 25 ml. of 3A- ethanol, treated with charcoal then filtered 0.05 ml. of hydrazine hydrate in 5 ml. of 3A-ethanol is added over a 15- minute period with stirring. The mixture is stirred 4 hours, treated with charcoal and filtered. The solution is evaporated to dryness and triturated with ether to give mg. of product; 4-hydrazono-l la-chloro-4-dedimethylaminotetracycline.

By reduction of this product in 3A-ethanol with 5 percent palladium on charcoal as catalyst and limiting the hydrogen uptake to l equivalent 4-hydrazono-44iedimethylaminotetracycline is produced.

Example Vll 5a,6-Anhydro-4-Hydrazono-4-Dedimethylaminotetracycline 4-Hydrazono-4-dedimethylaminotetracycline (3.4 g.) is dissolved in 68 ml. of methanol-concentrated hydrochloric acid (9:1) and the mixture refluxed for l minutes. The product which separates on cooling to room temperature is recovered by filtration and dried (2.0 g.).

The compound exhibits ultraviolet absorption maxima in 0.01 N methanolic-HCl at 268 and 423 mp. (typical 5a,6-anhydro absorption) plus additional absorption in the 300350 my. region (modified A-ring).

By means of this procedure the remaining products of example Vl are converted to their respective 5a,6-anhydro derivatives with the exception, of course, of the Ila-halo derivatives.

Example Vlll 4-l-lydroxyimino-4-Dedimethylaminotetracycline Partially purified 4-oxo-4-dedimethylaminotetracycline-4,6 -hemiketal g.) is dissolved in 60 ml. of methanol, stirred for minutes then filtered. To the filtrate there is added 30 g. of potassium bicarbonate an d 6.3 g. of hydroxylamine hydrochloride. The mixture is stirred 15 minutes at room temperature, filtered, and the filter washed with methanol. The combined filtrate and wash liquors are diluted with 200 ml. of water and the pH adjusted to 2.0 with concentrated hydrochloric acid. The crude product precipitates and is removed by filtration (6.6 g. Additional crude material (7.2 g.) is obtained by ether and ethyl acetate extractions of the mother liquor. The combined crude material is purified by dissolving in a mixture of toluene and isopropyl alcohol at the rate of l g. of crude in 3 ml. of toluene and 2 ml. of isopropyl alcohol, followed by immediate filtration. The pure material crystallizes upon standing and is dried at 50 C. in vacuo. 1! exhibits ultraviolet absorption maxima in 0.01 N methanolic- HCl at 309 and 360 mu.

Example IX 4-l-lydroxyimino-4-Dedmethylamino-5a,6-Anhydrotetracycline Crude 4-hydroxyimino-4-dedimethylaminotetracycline (500 mg. from example VIII is dissolved in 3 ml. of 4 percent methanolic-HCI the solution charcoaled and filtered. The charcoal filter cake is washed with methanol and the combined wash and mother liquors heated on a steam bath for 2-3 minutes, then allowed to stand at room temperature. The product which crystallizes on standing is recovered by filtration and dried in vacuo at room temperature. It exhibits typical anhydrotetracycline absorption in the ultraviolet region at 262 and 299 mp. in 0.01 N methanolic-HCI plus additional maxima at 422 mp. This latter maxima is due to the modified A-ring.

Analysis Found:

Calcd. for C, H,.N,O.-O.5CH,OH:

Example X Normal and Epi-4-Amind-4-Dedimethylamino-5a,6-Anhydrotetracycline Method A From 4-hydrazono-4-dedimethylaminotetracycline 4-Hydrazono-4-dedimethylaminotetracycline is slurried in 94 ml. of 1:1 solution of water and glacial acetic acid. Zinc dust (3.13 g.) is added and the mixture vigorously stirred at room temperature for 15 minutes. The mixture is filtered and the zinc cake washed with water-glacial acetic acid (lzl The filtrate is freeze-dried, the residue slurried with 100 ml. of water, filtered, and the solid product (zinc complex) dried in air to give a yield of 3.0 g.

The crude zinc complex is taken up in a hot mixture of 15 ml. tetrahydrofuran and 0.5 ml. concentrated hydrochloric acid. The solution is filtered hot and the solution allowed to cool whereupon the product crystallizes. It is isolated by filtration and dried in vacuo at 50 C. for 48 hours. The yield from 950 mg. of crude zinc complex is 400 mg. It exhibits ultraviolet absorption maxima at 223, 272 and 423 my. in 0.01 N methanolic-HCl, loge 4.45, 4.71 and 3.93, respectively.

The same compound can also be prepared from the purified amphoteric 4-aminotetracycline by the same process. Other acids and other solvents can be used in this process.

Method B From 4-hydroximino-4-dedimethylaminotetracycline A mixture of crude 4-hydroxylimino-4- dedimethylaminotetracycline (3.5 g.), zinc dust (3.5 g.) and 104 ml. of 50 percent aqueous acetic acid is stirred at room temperature for 15 minutes. The mixture is then filtered and the filter cake washed with 50 percent aqueous acetic acid. The combined wash liquor and mother liquor are diluted with an equal volume of water then freeze-dried. The residue is slurried with ether, filtered and dried, then slurried with 100 ml. water. The solid is recovered by filtration and air-dried to give 5.5 g. of crude zinc complex.

The zinc complex is decomposed to give the desired product according to the procedure of Method A.

Analysis Found- C.5h.7;H,5.l;N,$.5;Cl,

rum-1,, 3.2 Calcd. for c,,H,.o,N,Hc|- ci-|, ,0; c. 56.9; H, 5.4; N. 5.5 CI.

ExampleXl Method A 4-Norrnal and 4-epi-4-amino-4- dedimethylaminotetracycline To a slurry of 1.0 g. of 4-hydrazonov4- dedimethylaminotetracycline in ml. of 50 percent aqueous methanol at room temperature, 2.0 g. of sodium hydrosulfite is added and the mixture stirred one hour. More sodium hydrosulfite (1.0g.) is added and the stirring continued for an additional hour. A fourth gram of sodium hydrosulfite is then added and the stirring continued for another hour. The mixture is filtered, washed with 50 percent aqueous methanol and the filtrate evaporated in vacuo to remove the bulk of the methanol. The remaining aqueous solution is extracted with ethyl acetate to remove starting material and impurities of lower polarity than the desired product. The product is then extracted from the aqueous solution with n-butanol. The butanol extract is evaporated to dryness and the residue slurried with ether to give a solid product which is recovered by filtratron.

The crude product thus obtained is purified by taking 2.67 g. of product (the combined product from several such preparations) in ml. of dimethylformamide and adding 25 ml. of water. Crystallization begins immediately and after standing overnight at room temperature the product is recovered by filtration. It is washed with dimethylformamide until the wash liquor is colorless and then with ether. The yield is 2.0 g.

The product exhibits absorption maxima in the ultraviolet region in 0.01 N methanolic-HCI at 262 and 359 mp: E, Z2} of 340 and 272, respectively. Both the normal and 4-epi compounds, the latter is the predominant isomer, are detected on paper chromatography in the system nitromethaneztoluenezbutanol:pyridine (20: l0:5:3) as the mobile phase and Mcllvaine's buffer, pH 3.5. as the stationary phase.

Analysis Found: C, 55.8; H, 5.9; N. 9.2 Calcd. for C,,,H,,0.N, l.5(CH,),NCHO: C. 55.9; H. 5.9; N. 9.3

The products from both preparations show the typical tetracycline type ultraviolet absorption spectra in 0.0l N methanolic-HCl with maxima occurring at 260 and 360 my, log 425 and 4.17.

Method B From 4-hydroximino-4-dedimethylaminotetracycline Repetition of Method A but using 4-hydroxyimino-4- Example XII 4-Normal and cycline A. To a mixture of 4-hydroazono-4-dedimethylaminotetracycline (429 mg.), 01 ml. concentrated hydrochloric acid, 2 ml. dimethylformamide, 5 ml. water and 5 ml. glacial acetic acid is added 50 mg. of platinum oxide (prehydrogenated) in 5 ml. water and 5 ml. glacial acetic acid. The slurry is hydrogenated for 3 hours at room temperature and l atmosphere hydrogen, filtered and the solids washed with dimethylformamide. The combined washings and filtrate are stripped to dryness to give a mixture of 4-epi and 4-norrnalamino-4 dedimethylaminotetracyclines.

B. Repetition of this procedure but using only dimethyl formamide (no water or acetic acid) as solvent produces the same product.

C. The 4-hydrazono-l la-chloro-4-dedimethylaminotetracycline of example Vl-C is reduced with excess zinc in 50 percent aqueous acetic acid to give the zinc complex of the normal and epi forms of 4-amino-4- dedimethylaminotetracycline.

Example Xlll Example Xlll Isolation of Normal 4Amino-4-Dedimethylarninotetracycline Two hundred mg. of the mixture of 4-amino-4- dedimethylaminotetracyclines of example Xll-A are taken up in 5 ml. of 4 percent aqueous sodium bicarbonate and warmed on a steam bath for 30 minutes. The solution is cooled and adjusted to pH 4.5 with dilute hydrochloric acid. The acid solution is then extracted with ethyl acetate to remove the 4-epiamino-4-dedimethylaminotetracycline degradation products and then with n-butanol to extract the nonnal product. Evaporation of the n-butanol solution to dryness gives the desired 4-amino-4-dcdimethylaminotetracycline.

Example XIV The products of examples Vl-B, VII and IX are similarly reduced to a mixture of 4-epi and normal 4-amino-4- dedimethylaminotetracyclines by the procedures of examples Xl and XI]. In the case of the Sn, 6-anhydro compounds zincacetic acid is used as reducing agent.

The following compounds are thus prepared:

01 CH; OH

5 4-Epi-4-Amino-4-Dedimethylaminotetraem W;

EXAMPLE XV Reductive Amination of 4-0xo-4-Dedimethylaminotetracycline-4,6-Hemiketal A. To 300 mg. platinum oxide in 24 ml. of dimethylformamide (prereduced) is added a solution of 2.5 mg. of 4-oxo-4- dedimethylaminotetracycline-4,o-hemiketal and l.22 mg. of magnesium chloride hexahydrate in 36 ml. dimethylformamide. Ammonium hydroxide 3 ml. of 28 percent) is added and the mixture hydrogenated at room temperature and l atmosphere hydroogen for 3 hours. The catalyst is filtered off, washed with dimethylformamide and the combined wash and filtrate solutions added to 700 ml. ether with stirring. The solid filtered off, washed with ether and dried is the crude ammonium magnesium complex of 4-epi and 4-normal amino-4- dedimethylaminotetracycline.

The complex is broken up according to the procedure of example X to give the mixture of 4-amino-4-dedimethylamino-5 a,6-anhydrotetracyclines.

B. Repetition of this procedure using equimolar amounts of zinc, cadmium, cobalt, copper, nickel and manganese chlorides in place of magnesium chloride produces the same products.

Example XVl The procedure of example XV-A is repeated but using aqueous methylamine in place of ammonium hydroxide to give 4-methylamin0-4-dedimethylaminotetracycline consisting mainly of the 4-epi derivative with a small amount of the normal derivative.

Application of this procedure to the produces of example ll and using the appropriate primary amine produces a mixture of the 4-epi and nonnal derivatives of the following compounds in which the 4-epi form predominates.

H\ /Ri Y x 0H N I 1 --OH ()H I V sown, n I W ()H O i) Y X R,

H CH, C,H, H CH, My", H CH, i (,l-i, H CH, n-C-H,, H CH; l: H CH, cH,cH,orr H CH, CH,CH|CH,CH|OH H CH, cH,cHor-|cu, H CH, cH,cH,C.H. (:1 cu, cu, Cl CH,| CH,CH,OH Cl CH, n-CJ-l,

When the starting 4-oxo-4-dedimethylaminotetracycline- 4,6-hemiketal bears a 7-chloro group, rhodium rather than platinum oxide is preferred as catalyst.

Example XVII The reductive amination products of example XVI are transformed to their respective a,6 anhydro derivatives by the procedures of example IV.

Example XVIII tron Example XIX A mixture of crude 4-epi-4-amino-4-dedimethylaminotetracycline (500 mg. containing a small amount of the normal 4- amino compound), methyl iodide (2.5 ml.) and I0 ml. tetrahydrofuran is refluxed overnight. The mixture is evaporated to dryness to give a mixture of normal and 4-epi-4- methylamino-4-dedimethylaminotetracycline hydroiodide.

Repetition of this procedure but using ethyl, n-butyl, n-hexyl, benzyl, phenethyl, cyclohexyl, 2-hydroxyethyl, 2-hydroxypropyl and 4-hydroxy-butyl iodide in place of methyl iodide produces the corresponding 4-substituted amino-4- dedimethylaminotetracycline salts.

Example XX A mixture of 4-epiand 4-normal amino-4- dedimethylaminotetracycline (416 mg. 1 mM. and 2 mM. of each of ethyl iodide and propylene oxide is refluxed in 20 ml. acetone overnight. The mixture is evaporated to dryness, the residue washed with ether and dried. The product comprises a mixture of 4-epiand normal-4-diethylamino-4- dedimethylaminotetracyclinev Replacement of ethyl iodide by mbutyl, isopropyl and hexyl iodide produces the corresponding 4-dialkylamino-4- dedimethylaminotetracycline Example XXI The crude 4-epiand normal-4-methylamino-4- dedimethylaminotetracycline product of example XIX is taken up in water and the pH adjusted to 4.5. The acid solution is extracted with n-butanol and the butanol extract evaporated to dryness. The residue is then dissolved in ml. tetrahydrofuran, n-butyl iodide (2 ml.) added and the mixture refluxed overnight. Evaporation to dryness gives 4-N-(n-butyl), N-methyl-aminoA-dedimethylaminotetracycline hydroiodide (4-epiand normal configurations).

Example XXII Application of the procedures of examples XIX t'o XXI on the 4-amino-compounds of examples XIV produces the following products from the appropriate reactants.

The products are converted to the free base form by neutralization of the acid present and thence to various salts by the procedure of example XVIII. The following salts are prepared: hydrochloride, acetate, citrate, benzoate, sodium. calcium, manganese, aluminum and magnesium.

Example XXIII 4-0xo-l Ia-Chloro-G-Deoxy-6-DemethyI-6-Methylene-4- Dedimethylaminotetracycline 4-Oxo-l Ia-chloro-4-dedimethylaminotetracycline-4.6- hemiketal is dissolved in liquid hydrogen fluoride (in a ratio of 2 g./I5 ml.) at 0 C. After I5 minutes at this temperature the hydrogen fluoride is evaporated off to give the desired product.

In like manner, 4-oxo-6-deoxy-64lemethyl-6-methyIene-4- dedimethylaminotetracycline and 4-oxo-I Ia-fluoro-6-deoxy- 6-demethyl-6-methylene-4-dedimethylaminotetracycline are produced from 4-oxo-4-dedimethylaminotetracycline-4,6- hemiketal and 4-oxo-l Ia-fluoro-4-dedimethylaminotetracycline-4,6-hemiketal.

Example XXIV 4-l-Iydrazono-l Ia-Chloro-6 Deoxy-6-Demethyl-6-Methylene- 4-Dedimethylaminotetracycline Method A. A mixture of IO g. of 4-oxo-l la-chloro-6-deoxy- 6-dernethyl-6-methylene-4-dedimethylaminotetracycline, 6i ml. of methanol, 20 g. of potassium bicarbonate. and 3.8 g. of powdered hydrazine dihydrochloride is stirred for 30 minutes at room temperature. The reaction mixture is then filtered and washed with methanol. 900 ml. of water containing 6 ml. of concentrated hydrochloric acid is slowly added to the mother liquor with stirring and the mixture allowed to stand. The precipitated product is recovered by filtration, washed with water and dried.

Repetition of this procedure .but using hydroxylamine hydrochloride in place of hydrazine dihydrochloride produces 4-hydroxyimino-I I 6-deoxy-6-demethyl-6- methyIene-4-dedimethylaminotetracycline.

Method B. 4-Hydrazono-I Ia-chIoro-4- dedimethylaminotetracycline (product of example VI-C) is dissolved in liquid hydrofluoric acid at 0 C. (2 g./ l 5 ml. After l5 minutes at 0 C. the mixture is evaporated to dryness to give the title compound as the hydrofluoride salt.

The following compounds are prepared from appropriate reactants by the procedure of method A using the proper carbonyl group reagent in place of hydrazine dihydrochloride.

Example XXV 4-Epi and Normal 4-Amino-6-Deoxy-6-Demethyl-6- Methylene4-Dedimethylaminotetracycline The 4-hydrazono product of example XXIV (l g.) is slurried in 10 ml. of 50 percent aqueous acetic acid and zinc dust (2 g.) added. The mixture is stirred vigorously for 15 minutes then filtered and the filter cake washed with 50 percent acetic acid and water. The product, as zinc complex, is recovered by freeze drying the mother liquor.

The zinc complex is convened to the p-toluene sulfonate salt by treatment with excess p-toluene sulfonic acid in water. The p-toluene sulfonate salt is recovered by filtration and dried in vacuo.

Similarly, the 4-hydroxyimino analog of example XXIV is reduced to the same compound.

The free base is obtained by adjusting thepH of an aqueous solution of the p-toluene sulfonate salt to 5, filtering off and drying the product which separates.

The crystalline 5a,6-anhydr0-4-amino-4- dedimethylaminotetracycline is obtained when the crude zinc complex l g.) is refluxed in IS ml. of tetrahydrofuran containing 0.5 ml. concentrated HCl for 3 hours, then cooling to room temperature.

Similarly, the remaining products of example XXIV are reduced to give the title product.

Example XXVI The product of example XXIV as the base form is dissolved in methanol mg./5 ml.) and a freshly prepared solution of sodium hydrosulfite added (40 mg. in 5 ml. water). Afier minutes at room temperature the mixture is stripped of methanol and the residue extracted with n-butanol. Concentration of the n-butanol extract gives the 4-hydrazono-6- deoxy-6-demethyl-6-methylene-4-dedimethylaminotetracycline.

In like manner the remaining 1 la-halo products of example XXIV are converted to the corresponding deshalo compounds.

Example XXVII 4Hydroxy-6-Deoxy-6-Demethyl-6-Methylene-4- The 4-oxo product of example XXIII (l.0 g.) is dissolved in 12 ml. methanol and a freshly prepared solution of 0.8 g. sodiurn hydrosulfite in 20 ml. water added. After stirring 40 minutes at room temperature the mixture is combined with 10 ml. water and ml. ether. The ether extract is separated, the aqueous layer reextracted with another 50 ml. ether and the combined ethereal solutions dried over sodium sulfate. Evaporation to dryness gives the crude product.

In like manner the remaining 4-oxo compounds of example XXIII are reduced to the title compound.

Example XXVIII The product of example XXV (500 mg. is refluxed with methyl iodide (2.5 ml.) in tetrahydrofuran (10 ml.) for 15 hours. Evaporation of the mixture gives a mixture of normal and 4-epi4-methylamino-6-deoxy-6-demethyl-6- methylenetetracycline hydroiodide.

Repetition of this procedure but using ethyl, n-butyl, n-hexyl, benzyl, phenethyl, cyclohexyl, Z-hydroxethyl, Z-hydroxypropyl and 4-hydroxybutyl iodide in place of methyl iodide produces the corresponding 4-substituted amino-4- dedimethylaminotetracycline salts.

Example XXIX Example XXX The crude 4-epiand nonnal-4-methylamino-6deoxy-6- demethyl--methylene4-dedimethylaminotetracycline product of example XXVIII is taken up in water and the pH adjusted to 4.5. The acid solution is extracted with n-butanol and the butanol extract evaporated to dryness. The residue is then dissolved in 10 ml. tetrahydrofuran, n-butyl iodide (2 ml.) added and the mixture refluxed overnight. Evaporation to dryness gives 4-N-( n-butyl N-methyl-amino-4- dedimethylaminotetracycline hydroiodide (4-epi-and normal configurations).

, Example XXXI The sodium, potassium calcium, barium, aluminum and magnesium salts of the products of examples XXIII-XXX are prepared by the procedure of example XVIII.

The acid salts, e.g. hydrochloride, sulfate. citrate, gluconate, citrate and benzoate, of the products of example XXV, XXVIII-XXX are prepared according to the procedure of example XVIII.

What is claimed is:

and the alkali metal and alkaline earth metal salts thereof wherein Z is selected from the group consisting of hydrogen, chloro and fluoro; X is selected from the group consisting of methyl and hydrogen, with the proviso that X is methyl when 2 is hydrogen; and R is selected from the group consisting of OH, NHCONl-h, NHC(NH)NH,, NHCSNH,, 4(,B-hydroxyethyl)piperazino, and NR R wherein R is selected from the group consisting of hydrogen, alkyl of from one to six carbon atoms, phenyl, benzyl and cyclohexyl and R is selected from the group consisting of hydrogen, alkyl of from one to six carbon atoms, phenyl and benzyl.

2. The compound of claim 1 wherein X is hydrogen, Z is fluoro and R, is --OH.

3. The compound of claim 1 wherein X is methyl, 2 is hydrogen and R is NR R wherein R and R, are hydrogen.

4. A compound of the formula 4. A compound of the formula 333 UNITED STATES PATENT OFFICE 1 CERTIFICATE OF CORRECTION Patent. No. h Dated November 3: 97

B obert K. Blackwood and Charles E. Stephens Jr.

It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Col. 1, lines 60-69, the second formula reading Y H 0 0H NRlR2 Y H 6 OH NR R should OH OH read I H -CONH CONH I OH O O O OH O 0 Col. 2, lines 53-62, the formulae reading m3 OH 0 OH OH 001m 001m OH O O O I II 3 0 shggld read 3 OH ONH Col, 3, line 66, under the left-hand formula, should read XI g-333 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION 3, 7 Page 2 Patent No. Dated It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Col. 5, lines 3 4- HI, the formula reading CONH2 r should read O=C I OH 0 o CONHQ lines 19-58, the formula reading Y x 0 N( H3) Y x 0 M 3 H should H QHI read ONH2 ON OH 0 Z OH O OH 0 Z H C01. 9, line 57, "5 77" ShOuld read 5.77 --3 line 62, "3 7" should read 3.7 line 67, "2 2" should read 2.2

Col. 10, line 1, 632, 655" should read 6.32, 6.55

line 55, "mg." should read Mg 75,-1.3? UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. Dated Page 3 Invent or s -i It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

601. 18, lines 10-16, the formula reading j Y x R132 Y X M1 11 \-OH H 1 CR OH CONH should read" H I ONH--, /Y 2 2 CH on 0 b on OH 0 0 line 69, "ll6-deoxy" should read l1a-ch1oro-6-deoxy Col. 19, lines 4-1 4, the formula reading 5* W3 CH2 NR3 H should OH H IEg read com:

OH 0 Z O 0 AH 0 Z 0 A Col. 20, line 61 et. seq., table should be read as though it appears under formula in Column 19. Also, the second R variable of the second column of this table reading "(013 1 should read N(CH lines 1-9, the formula reading X OH NR3 X H 3 H OH H I CONH o 0 Z 0 N Col 22, line 7, delete "4. A compound of the formula".

Signed and sealed this 13th day of June 1972,

(SEAL) Attest:

flglgggl v gmg rmmm. ROBERT GOTTSCHALK 3 car Commissioner of Patents 

2. The compound of claim 1 wherein X is hydrogen, Z is fluoro and R3 is -OH.
 3. The compound of claim 1 wherein X is methyl, Z is hydrogen and R3 is NR4R5 wherein R4 and R5 are hydrogen.
 4. A compound of the formula
 5. 4-dedimethylamino-4-oxo-tetracycline-4-methylhydrazone. 